Microfluidic devices for detecting and characterizing analytes, such as cells, in a sample fluid - Patents.com

Abstract

An apparatus for detecting an analyte, such as a cell, in a sample fluid, the apparatus comprising: a fluid channel having a longitudinal channel axis and configured to allow passage of a sample fluid containing the analyte; at least one pair of electrodes; an electric field generating unit for generating an electric field between the at least one pair of electrodes; and a detection unit for detecting the analyte in the sample fluid passing through the electric field. Each electrode of the at least one pair of electrodes has a three-dimensionally structured electrode surface. The electrode surface is curved with respect to the longitudinal channel axis. The apparatus may further comprise an optical sensing unit structured to optically sense a sensing region of the fluid channel between the at least one pair of electrodes.

Description

[Technical field]

[0001] The present disclosure relates to microfluidic devices for detecting and characterizing analytes, such as cells, in a sample fluid. [Background technology]

[0002] Existing microfluidic devices employing thin-film electrodes for electrical impedance measurements for analyte (particle or cell) identification rely on flat electrodes placed on the bottom or top side of the microfluidic channel. Such configurations have low sensitivity because the analytes to be detected, e.g. particles or (blood) cells, are partially covered by a non-uniform electric field. The signal quality then depends on the position of the particle in the channel. In some cases, the analytes are completely covered by a uniform electric field. This leads to high noise levels, which hinders reliable particle detection and characterization. This reduces sensitivity and also the signal-to-noise ratio. Summary of the Invention [Problem to be solved by the invention]

[0003] In view of the above, it is an object of the present disclosure to provide an improved microfluidic device with higher sensitivity and higher signal-to-noise ratio. [Means for solving the problem]

[0004] According to a first embodiment of the present disclosure, an apparatus for detecting an analyte, such as a cell, in a sample fluid comprises a fluid channel having a longitudinal channel axis and configured to allow passage of a sample fluid containing the analyte, at least one pair of electrodes, an electric field generating unit for generating an electric field between the at least one pair of electrodes, and a detection unit for detecting the analyte in the sample fluid passing through the electric field. Each electrode of the at least one pair of electrodes has a three-dimensionally structured electrode surface. The electrode surface is curved with respect to the longitudinal channel axis.

[0005] The curved surface of the electrodes surrounding the longitudinal channel axis provides better coverage of the sensing area of ​​the fluid channel between the electrodes, and because the curved surface is located near or around the longitudinal channel axis, it provides higher sensitivity and improved signal-to-noise ratio.

[0006] According to one embodiment of the present disclosure, the curved electrode surface of each electrode of the at least one pair of electrodes has a concave electrode surface that partially surrounds a sensing region between the electrodes and is three-dimensionally disposed near or around the longitudinal channel axis, thereby improving sensitivity and therefore improving sensitivity to sensing an analyte passing through the sensing region of the fluid channel between the electrodes.

[0007] The sensitivity of at least one electrode can be adjusted or improved for its sensing application by adjusting the arc length of each of the concentrically oriented electrodes, in one example, the arc length is between 10° and 135°, such as between 10° and 60°, particularly between 10° and 90°.

[0008] In other embodiments of the present disclosure, the fluid channel has a cross-section that includes a circular or elliptical shape.

[0009] In a further beneficial, but non-limiting configuration, the electrodes of at least one pair of electrodes are configured on opposite sides of the fluid channel and face each other.

[0010] A further improved embodiment of the present disclosure comprises an optical sensing unit for optically sensing the sensing area of ​​the fluid channel between at least one pair of electrodes. With this embodiment, an apparatus (highly sensitive microfluidic device) according to the present disclosure is designed to electrically and optically detect analytes such as particles or cells in an electrolyte solution. Multi / multi-frequency electrical impedance measurements using the electric field generating unit provide information about the dielectric properties, its internal and external morphology, and size of the detected analytes.

[0011] Additional optical data obtained by the optical sensing unit provides information about physical properties such as internal and external morphology and size. By combining these electrical and optical data sets, the device according to the present disclosure can be used as a flow cytometer for classification, enumeration, and extraction of analytes or target particles in suspension, such as biological cells in blood. The device according to the present disclosure can also be used as a hematology analyzer for classification and counting of blood cells. In addition to blood cell counting, detection of rare cell populations circulating in the blood for disease diagnosis and prognosis is also possible.

[0012] In one embodiment, the optical detection unit has an optical sensing axis disposed at the mid-plane of the fluid channel, the optical sensing axis extending perpendicular to a longitudinal channel axis of the fluid channel, and may further comprise a photodetector offset from the mid-plane of the fluid channel.

[0013] In an embodiment, the optical detection unit comprises a waveguide unit, a laser device, or an optical sensor, for example a charge coupled device (CCD), a CMOS device, or the like.

[0014] In the device according to the present disclosure, at least one pair of electrodes does not obstruct the optical path of the light-sensing unit for illumination and optical sensing, which leads to a particularly strong improvement in sensitivity.

[0015] In a further example, the device according to the present disclosure comprises a first substrate and a second substrate. The first substrate and the second substrate at least partially form a fluidic channel. Furthermore, the device may further comprise at least one integrated waveguide unit optically coupled to a sensing region of the fluidic channel between at least one pair of electrodes. For example, the at least one waveguide unit may be disposed between the first substrate and the second substrate and / or integrated into the first substrate or the second substrate.

[0016] Alternatively, the first substrate may have a first recess and the second substrate may have a second recess, the first recess and the second recess at least partially forming the fluid channel. In an advantageous embodiment, the first recess and the second recess have the same shape. In a further embodiment, the first recess and the second recess may each include an electrode disposed therein.

[0017] To improve the sensitivity of the device, the first electrode may have substantially the same shape as the second electrode. One or more of the electrodes may have a surface with a cross-section that includes a circular shape. Alternatively, one or more of the electrodes may have a surface with a cross-section that includes an elliptical shape.

[0018] In a further example, the detection unit may detect the analyte in the sample fluid with a voltage drop between the electrodes in the range of 0.1-0.5V, preferably 0.2V-0.4V. [Brief description of the drawings]

[0019] The present disclosure will now be described with reference to the drawings. [Figure 1] 1a-1c show an example of a microfluidic device in the art that implements a planar electrode layout. [Diagram 2] FIG. 1 shows another example of a microfluidic device in the art that implements a parallel opposed planar electrode layout. [Diagram 3] FIG. 1 illustrates a known application of the Coulter Principle in a known blood analyzer. [Figure 4] FIG. 1 illustrates the VCS principle for blood cell analysis. [Diagram 5] FIG. 1 is a diagram showing an example of the operating principle of a flow cytometer. [Figure 6] 1A and 1B are schematic diagrams illustrating an example of a microfluidic device according to the present disclosure and a measurement method according to the present disclosure. [Figure 7] FIG. 7 shows further details of the example of FIG. 6. [Figure 8]8a-8d show the electric field distribution for several electrode configurations. [Figure 9] 9a and 9b show the extraction of the electrical impedance signal in a whole cell measurement. [Figure 10] 10a and 10b show the results of the extracted electrical impedance signal in a nucleus-only measurement. [Figure 11] 11a and 11b show another example of a microfluidic device according to the present disclosure. [Figure 12] FIG. 1 shows a scatter plot of the results of a three-parameter blood cell count performed using one of the versions of the device defined in the present disclosure. [Figure 13] FIG. 1 shows differences in electrical properties (opacity distribution) of breast cancer tumor cell lines (MCF-7 and MDA-MB-231) and blood cells. [Figure 14] 14a and 14b show the results of drug response analysis on cells of a breast cancer cell line (MCF-7). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] In the following detailed description, for a proper understanding of the present disclosure, corresponding elements or portions of the present disclosure are designated by the same reference numerals in the drawings.

[0021] The present disclosure provides microfluidic devices with integrated electrodes as sensors for sensitive combined electrical and optical detection, identification and / or characterization of particles (analytes) in electrolyte solutions. Multi / multi-frequency electrical impedance measurements can provide information on the dielectric properties of the particles (analytes), as well as the internal and external morphology of the particles (analytes), and the size, shape and / or phenotype of the particles (analytes). Optical data can provide information on the internal and external morphology, optical (refractive and transmissive) properties related to the size, shape and / or phenotype of the analytes. Combining these rich data sets, the present disclosure can be used as a flow cytometer for detection, identification, enumeration, classification and characterization of target particles (analytes) in suspension, such as biological cells in blood.

[0022] The present disclosure can also be used as a hematology analyzer for blood cell differentiation and counting. In addition to blood cell counting, detection of rare cell populations circulating in the blood or other secretions from these cells, such as exosomes, is also possible for disease diagnosis and prognosis.

[0023] The detection, identification, enumeration, classification and / or characterization of any kind of analyte, such as particles, cells, vesicles, bacteria, etc., requires processing at the single analyte (particle or cell) level. For this reason, microfluidic tools are needed at these length scales. Prior art microfluidic devices that successfully employ electrical impedance measurements for analyte, particle or cell differentiation generally rely on a planar electrode configuration. This configuration has been developed in two known versions:

[0024] 1a-1c depict an example of such a known microfluidic device 10 according to the state of the art implementing a planar electrode layout. As shown in FIG. 1a, a number of planar electrodes 12-1, 12-2, 12-3 are constructed or arranged at the bottom of a channel 11. In this example, the three electrodes are arranged along the flow direction of the channel 11. An excitation signal 13 is applied to the central electrode 12-1, and the difference in electrical impedance of the side electrodes 12-2, 12-3 located upstream and downstream from the central electrode 12-1 is measured and output via an electronic circuit.

[0025] Although this configuration is easy to manufacture, the particles (indicated by the numbers 1, 2, 3 in FIG. 1a) are partially covered by the electric field formed in the channel 11. The electric field is not uniform, which leads to very low sensitivity and signal-to-noise ratio. FIG. 1b shows the magnitude of a typical electric field distribution in the channel 11. Due to this non-homogeneous electric field generated by known configurations of electric field generating units (not shown), the signal quality is highly dependent on the height position of the particles in the channel relative to the bottom of the channel 11 or to the planar electrodes 12-1, 12-2, 12-3 placed at the bottom.

[0026] Figure 1c shows a typical differential signal output generated by a particle (number 1, 2 or 3) passing through the sensing region in the channel 11 at three different heights. As seen in Figure 1, a differential layout is applied to reduce noise and improve performance by using a pair of electrodes as a reference and subtracting the drift and noise of the signal. However, the sensitivity never reaches the level required for any biomedical or analytical application. Although this known configuration is easy and cheap to manufacture, making it suitable for scale-up and industrialization, its poor performance makes it not an option.

[0027] Figure 2 shows a high-performance version of the state-of-the-art planar electrode layout, where two parallel counter electrodes 12-1a, 12-1b and 12-2a, 12-2b are arranged (attached or placed) on the upper segment 11a and the lower segment 11b of the channel 11. This arrangement provides a uniform electric field distribution, improving the signal-to-noise ratio and sensitivity. It is the best performing option reported in the literature so far, and provides the sensitivity required for biomedical applications such as the detection of biological cells.

[0028] However, such devices are hindered from scale-up and industrialization of their fabrication because they require an intermediate dielectric layer to which the top and bottom planar substrate layers are attached. Therefore, this configuration can only be realized in a laboratory environment. In addition to this problem, another important drawback of this configuration is that the electrodes block optical access to the measurement volume.

[0029] The signal quality is still influenced by the particle's position, being higher when the particle is closer to one of the electrodes (top or bottom). However, this is not as dominant as in the previous case with planar electrodes. The noise level of this improved configuration is still too high for accurate measurements. For this reason, a differential measurement approach with a dual electrode pair was attempted to further reduce the noise level. However, the sensitivity level is still limited to single particle or cell-level events, limiting the characterization possibilities.

[0030] The basic principle of the measurement approach of this disclosure is based on the Coulter Principle. The Coulter Principle is based on counting blood cells by measuring the change in electrical conductance as cells suspended in a conductive fluid pass through a small orifice. Today, more than 98% of automated cell counters (hematology analyzers) incorporate this technology, which is called the "Coulter Principle". Figure 3 shows how the Coulter Principle works in a modern hematology analyzer.

[0031] Another variation of this measurement technique was introduced by Coulter more than 20 years ago and is called the “Volume-Conductivity-Scattering (VCS) technique” shown in Figure 4. Cells are passed through an aperture to which an alternating voltage is applied. Direct current (DC) and radio frequency (RF) impedance measurements are combined with light scattering measurements. The DC data are used to normalize the RF data, removing the effect of particle position on the signal amplitude. Variants of the Coulter principle are used to distinguish and enumerate cell populations in blood. Since each population possesses a very large number of cells (from thousands to millions), and coincidence of measurements, in other words overlapping peaks, occurs frequently, a post-quantification process based on statistical inference is used. Therefore, this method lacks the specificity to identify rare cell populations in blood.

[0032] Prior art flow cytometers are different types of particle analysis devices based on optical measurements. Typically, particles are first hydrodynamically focused in a stream, then excited with a focused laser light, and the scattered light at different angles is collected. Particles can also be stained with fluorescent markers to obtain specific information. Flow cytometers provide fast and robust analysis, but require at least 104 particle / cell events for population detection and classification. This makes flow cytometers ineffective for rare particle / cell populations (<10 cells in 1 mL of liquid). Moreover, flow cytometers require the particles / cells to be detected in suspension to be stained with optical biomarkers. Thus, the performance of a flow cytometer is always limited to the biomarker panel selected by the user.

[0033] The working principle of a flow cytometer is shown in Figure 5. Although flow cytometers are fast and reliable, they are very expensive, complex and bulky systems. Successful application of flow cytometers requires laborious sample preparation, trained staff and the use of specific antibodies, limiting their use in blood analysis.

[0034] The present disclosure employs microfluidic technology utilizing a three-dimensional electrode configuration to enable simultaneous sensitive electrical and optical measurements of analytes such as cells / particles. In some embodiments of the present disclosure, this combined measurement approach can be performed in a label-free manner, where multi-frequency electrical impedance measurements provide dielectric signatures about the internal and external morphology, size, shape, and / or phenotype of analytes such as cells / particles, as well as using biomarkers to provide data about specific properties set by the user.

[0035] This disclosure is based on a new microfluidic device design with an improved three-dimensional electrode configuration that provides high sensitivity and signal-to-noise ratio, and can be tuned to the desired size range of particles for improved performance.

[0036] 6, 7a, 7b, and 7c show non-limiting schematic examples of configurations of a microfluidic device 100 according to the present disclosure. The microfluidic device 100 according to the present disclosure enables multi-frequency electrical impedance measurements of analytes 1, 2, 3 (such as blood cells) passing through a fluidic channel 110 having a longitudinal channel axis 110z using a pair of three-dimensional curved first and second electrodes 120a and 120b centered about the longitudinal channel axis 110z. As shown in the figures, the first and second electrodes 120a and 120b have three-dimensional structured electrode surfaces positioned with respect to the longitudinal channel axis 110z, thereby at least partially surrounding the fluidic channel 110.

[0037] The microfluidic device 100 may include a first substrate 130a and a second substrate 130b. Suitable materials for manufacturing the one or more substrates 130a, 130b preferably include transparent materials compatible with microfabrication and thin film processing techniques, such as glass and silica. At least a portion of the wall of the fluidic channel 110 may be formed by a first recess 131a in the first substrate 130a. Additionally or alternatively, at least a portion of the wall of the fluidic channel 110 may be formed by a second recess 131b in the second substrate 130b. Preferably, the first recess 131a has the same shape as the second recess 131b. This significantly simplifies the manufacturing of the microfluidic device. Most preferably, the first recess 131a and the second recess 131b are symmetrical with respect to a mid-plane or central plane 110z of the fluidic channel 110.

[0038] The electrodes 120a, 120b may be disposed opposite each other on different, preferably opposite, sides of the fluidic channel 110. The first electrode 120a may be disposed in a recess 131a of the first substrate 130a. The second electrode 120b may be disposed in a recess 131b of the second substrate 130b. Alternatively, one or more three-dimensionally curved electrodes of the present disclosure, such as the electrodes 120a, 120b, may be disposed in another selected location such that the electrodes face each other, or may be disposed in another manner consistent with the principles of the present disclosure. Examples of processes that can be used to dispose the electrodes include, but are not limited to, deposition, sputtering, or direct printing of metal, etc. In the present disclosure, the term "disposed" shall be construed to include within its meaning any type of deposition, disposition, attachment, integration, or other manner of providing one or more electrodes, in any other manner consistent with the present disclosure.

[0039] Reference numeral 140 generally indicates the configuration of an electric field generating unit, which is connected to both electrodes 120a, 120b and is capable of applying an electric field distribution in the channel 110 between both electrodes 120a, 120b. Electric field lines 141 generally indicate the electric field distribution in the channel 110 between both electrodes 120a and 120b.

[0040] The microfluidic device 100 according to the present disclosure may further include an optical sensing unit 150 (see FIG. 6 and FIG. 7C) arranged to optically detect or sense a sensing region of the fluidic channel 110 between at least a pair of the first electrode 120a and the second electrode 120b. The optical sensing unit 150 may be configured as or include an optical waveguide, a laser device, and / or an optical sensor. In another useful example, the optical sensor may be a charge-coupled device (CCD), a CMOS device, etc. In one embodiment of the present disclosure, the optical measurement using the optical sensing unit 150 is performed simultaneously (preferably synchronously) with the electrical measurement, for example, using the electrodes 120a and 120b. The electrodes 120a, 120b are arranged on only a portion of the inner wall 110′ of the fluidic channel 110, so that they do not obstruct the optical sensing axis 150a of the optical sensing unit 150. Thus, label-free (LED illumination, multi-focal or multi-depth placement, transmitted or reflected light, lens-free imaging, waveguide integration, PMT integration, etc.) or fluorescent (using specific biomarkers for staining) measurements are possible. In one exemplary microfluidic device 100, the optical sensing axis 150a may be perpendicular to the flow direction through the fluidic channel 110, but may be horizontal in other exemplary embodiments. In general, the optical sensing axis 150a may form an angle with respect to the direction of flow through the fluidic channel 110 to achieve optical measurements through the portion(s) of the first and second substrates 130a and 130b that are not obstructed by the electrodes 120a and 120b.

[0041] 7A, 7B, and 7C show several side views of a microfluidic device 100 according to the present disclosure having a pair of first and second electrodes 120a and 120b and configurations of electric field lines. The pair of electrodes 120a, 120b face each other from different (preferably opposite) sides of the fluidic channel 110. The first and second electrodes 120a and 120b have three-dimensional structured electrode surfaces centered about the longitudinal channel axis 110z, thereby at least partially surrounding the fluidic channel 110. In some exemplary embodiments, one or more of the electrodes are oriented at least partially concentrically with respect to the longitudinal channel axis of the fluidic channel 110. In exemplary embodiments, the electrodes 120a and 120b are three-dimensionally curved electrodes, preferably concave. The concave surface of the first electrode 120a preferably faces the concave surface of the second electrode 120b. One or more of the three-dimensionally curved electrodes may have at least partially cylindrical surfaces. Preferably, one or more of the electrodes has a surface with a cross-section that comprises at least a partly circular or partly elliptical shape, and / or is circular or elliptical in shape.

[0042] As shown in Figure 2, the electrode configuration according to the present disclosure provides improved performance compared to known prior art devices that implement planar electrodes arranged in parallel opposing configurations above and below a channel. Such prior art device configurations prevent optical visibility and therefore cannot be coupled to an optical system for real-time coupling for measurements as disclosed herein. In contrast, embodiments of the microfluidic device 100 according to the present disclosure not only provide optical visibility but also allow the fabrication of multiple depth channels on a single chip / device for higher performance.

[0043] In the exemplary embodiment shown in Figures 6 and 7, the arc length of each electrode 120a, 120b is approximately 90° when viewed along the curvature of the curved surface of the electrode 120a, 120b. However, in other exemplary embodiments, the electrodes 120a, 120b may be implemented to have an arc length between 10° and 135°, such as between 10° and 60° or between 10° and 90° when viewed along the curvature of the curved surface of the electrode 120a, 120b. As shown in Figures 6 and 7, the arc length of each electrode 120a, 120b is approximately 90°. In the illustrated example, the fluid channel 110 has an elliptical cross-section, although a circular cross-section is equally feasible. In an exemplary embodiment, the fluid channel 110 may have an inner wall 110' that is at least partially cylindrical. Preferably, the fluid channel 110 has an at least partially circular or partially elliptical cross-section and / or includes an inner wall 11' that is circular or elliptical in shape.

[0044] The performance of the microfluidic device according to the present disclosure was compared with the known prior art implementing top-bottom planar electrodes as shown in FIG. 2. Numerical studies were performed to determine the frequency-dependent response of a cell with a diameter of 12 μm and a nucleus-cytoplasmic ratio of 0.5 in 1X PBS buffer. The electrical parameters of the cell were typical and extracted from the literature. The dimensions of the measurement volume were a square of 20 μm x 20 μm for the top-bottom electrode arrangement and a circular shape of 20 μm diameter for the new three-dimensional concave electrode arrangement. For a fair comparison, the penetration rate of the electrodes into the microchannel was set between 10% and 100% and the data were compared.

[0045] 8a and 8d show typical arrangements and distribution of electric field lines for the three-dimensional electrode arrangement of the microfluidic device 100 according to the present disclosure (10% in FIG. 8a, 50% in FIG. 8b) and the top-bottom electrode arrangement of the prior art (FIG. 2) (20% in FIG. 8c, 100% in FIG. 8d). The legends and flow lines represent the electric field strength and distribution, respectively.

[0046] 9a and 9b show the results of electrical impedance signals (frequency dependent impedance response) extracted in whole cell measurements for different electrode configurations. Fig. 9a relates to measurements made with the three-dimensional geometry electrode configuration of the microfluidic device 100 according to the present disclosure. Fig. 9b relates to measurements made with the prior art top-bottom electrode configuration as shown in Fig. 2.

[0047] Similarly, Figures 10a and 10b show the results of extracted electrical impedance signals (frequency dependent impedance response) for nuclei only measurements. Figure 10a relates to measurements performed with a three-dimensional geometry electrode configuration of a microfluidic device according to the present disclosure. Figure 10b relates to measurements performed with a prior art top-bottom electrode configuration as shown in Figure 2.

[0048] The embodiments of the microfluidic device according to the present disclosure provide a higher span over the entire frequency range and typically provide higher amplitudes compared to the respective prior art configurations. One important feature of the new electrode configuration is that at frequencies below 1 MHz, for example, the overlapping signals can distinguish the contents inside cells, which was not possible with the top-bottom configuration, as visualized in FIG. 10b.

[0049] 11a and 11b show schematic non-limiting examples of other aspects of the microfluidic devices 100' and 100'' according to the present disclosure. Such exemplary embodiments comprise integrated optics for more advanced and high performance applications. An integrated waveguide unit, e.g. 160a, 160b, is optically coupled to a sensing region of the fluidic channel between at least two electrodes 120a and 120b. The exemplary microfluidic devices 100', 100'' illustrated in FIG. 11a and FIG. 11b may include a first substrate 130a and a second substrate 130b. The fluidic channel 110 of the exemplary embodiments 100' and 100'' may be formed, at least in part, by a recess 131a in the substrate 130a and a recess 131b in the substrate 130b.

[0050] FIG. 11a showing an example 100′ illustrates an exemplary embodiment of an integrated optical system utilizing waveguide units 160a and 160b. One or more waveguide units, e.g., 160a, 160b, may be disposed in an intermediate region between substrates 130a and 130b, generally designated by a mid-plane or mid-planar surface 160c. In this exemplary embodiment, the waveguide units 160a, 160b are disposed between a first substrate 130a and a second substrate 130b, preferably with the substrates 130a and 130b glued or bonded to each other. The material of the waveguide is selected based on the required refractive index difference compared to the substrate material associated with the intended application. This difference defines how light is trapped and guided. Waveguide units suitable for use in embodiments of the present disclosure can be made, for example, by depositing / coating a material (e.g., silica or a polymer material) onto the substrates 130a and 130b and patterning it using microfabrication techniques similar to those used to fabricate the substrates. As an alternative to an integrated waveguide unit or units, the focused laser beam from laser device 180 can be aligned with mid-plane 160c as an external component serving the same purpose, resulting in easier and cheaper manufacture, albeit with reduced performance.

[0051] FIG. 11B shows an alternative configuration, designated by reference numeral 100″. The waveguide units 160a and 160b may be disposed on the lower surface 110b and the upper surface 110a of the channel 110, respectively. The waveguide units may be disposed on or within the substrates 130a and 130b. In one embodiment, the optical axis of one or more of the waveguide units 160a and 160b is disposed along a plane 160d. The plane 160d is oriented at a non-zero angle, preferably generally orthogonal to the mid-plane 160c or central plane 160c. In this configuration, the waveguides need to be integrated after forming the recesses 131a and 131b in the substrates 130a and 130b.

[0052] The fabrication process and material options can be the same or similar to those described in connection with the previous configurations shown in Figures 6, 7a, 7c and 11a. The material may be deposited on a template or deposited and then patterned to form the waveguide. The thickness of the waveguide is about two orders of magnitude smaller than the depth of the fluidic channel 110, so it may be placed on the surface of the fluidic channel 110. In this case, the flow path is not obstructed by the waveguide unit. For shallow channels where the thickness of the waveguide would cause disturbances in the flow field, a recess can be made by removing material from the substrate after the waveguide is placed to obtain a horizontal surface of the channel 110.

[0053] As an alternative to an integrated waveguide, far-field optics can be used in this configuration. Optical components such as a light source to focus light and an image sensor to focus and detect scattered light can be located at the top and bottom of the device (externally). The waveguide units 160a and 160b of both embodiments 100' and 100'' of Fig. 11A and Fig. 11B are optically coupled to the detection region of the fluidic channel between at least two electrodes 120a and 120b, providing an additional integrated optical path that allows illumination of analytes 1, 2, 3 (particles or cells) by a (focused) light source and collection of scattered light in different planes, optionally with several angles, in combination with a detector 170 to analyze the analytes. The detector 170 may be located at a position off the optical path 160c or 160d of the waveguide units 160a, 160b. In such an embodiment of the present disclosure, the optical axis of the detector forms a non-zero angle with the optical axis of the waveguide. By using external optics as an alternative or in combination with integrated waveguides, more functionality can be added to the system, reducing the overall cost.

[0054] In a first embodiment shown in Fig. 11A, when light leaving the waveguide unit 160a or a focused light (laser) beam 180 located at the mid-plane 160c of the channel 110 illuminates the analytes (cells or particles) 1, 2, 3, forward scattering (FSC) and side scattering (SSC) are generated, which can be read, detected or sensed by the optical sensing unit 150 including the waveguide unit 160b and the detector 170, respectively. The detector 170 may be located at a position off the optical path 160c of the waveguide units 160a and 160b. The detector 170 may be directly attached to the device 100'-100'' or may be located at a distance based on the requirements of the optical sensing application or the location of the optical setup or peripheral components.

[0055] In a second embodiment, as shown in Fig. 11b, the waveguide units 160a and 160b are integrated into the top surface 110a and / or the bottom surface 110b of the channel 110 for illumination and collection of forward and back scattered light. The waveguide unit 160a illuminates the analytes 1, 2, 3 and collects the back scattered light, and the waveguide unit 160b collects the forward scattered light. The second option is more suitable for shallow fluidic channels 110. In some embodiments of the present disclosure, the first waveguide unit 160a and / or the second waveguide unit 160b are integrated into the first substrate 130a and / or the second substrate 130b.

[0056] Examples of microfluidic devices 100, 100' and 100'' according to the present disclosure implement three-dimensional curved electrode configurations. The three-dimensional structured electrode surfaces can be advantageously arranged about the longitudinal channel axis, thereby at least partially surrounding the fluid channel. In some exemplary embodiments, the configurations include a pair of electrodes oriented at least partially concentrically. Some configurations utilize or implement a pair of concave electrodes. Microfluidic devices according to the present disclosure achieve higher sensitivity compared to known prior art devices. Measurements are further improved by performing simultaneous (preferably synchronized) electrical and optical measurements, while integration of optical waveguides to create a hybrid Coulter principle particle (hematology) analyzer and flow cytometer achieves even improved sensitivity and specificity levels.

[0057] Exemplary microfluidic devices 100-100'-100'' according to the present disclosure can achieve high sensitivity and specificity levels in electrical measurements for identifying analytes such as normal blood cells, tumor cells and cell clusters, or for analyzing drug response of tumor cells. Exemplary microfluidic devices according to the present disclosure can be used as sensor devices for liquid biopsies such as rare cell detection in blood, hematology analyzers, microscale flow cytometers, and the like.

[0058] FIG. 12 represents a three-parameter blood cell count performed using one of the versions of the device defined in this disclosure. Three major subgroups of blood cells, namely neutrophils, lymphocytes, and monocytes, were identified in proportions of 71%, 23%, and 6%, respectively, for a healthy adult donor. The obtained proportions were consistent with typical blood cell counts in healthy adults. The measurement was performed completely label-free, without any sample preparation, using multi / multi-frequency electrical impedance measurements on diluted blood samples (diluted in 1X PBS buffer). All measured frequencies were used to define clusters of blood cells, and cellular events detected at only one frequency were selected for illustration (FIG. 12). Although more advanced blood cell counts are possible with modern hematology analyzers, the ability to count immune cells during liquid biopsy applications could provide additional data for better decision-making for cancer treatment, since the number of immune cells is associated with disease progression.

[0059] Figure 13 shows histogram plots of opacity distribution for breast cancer tumor cell lines (MCF-7 and MDA-MB-231) and blood cells. Opacity is defined as the ratio of high-frequency data to low-frequency data. These high- and low-frequency values ​​vary depending on the design and application. In this particular example, breast cancer tumor cells and blood cells could be distinguished with a high success rate. Furthermore, the difference in the characteristics of MCF-7 cells and MDA-MB-231 cells is also clear. Therefore, it is also possible to distinguish different cell types of the same origin, such as different tumor cells of the same cancer type.

[0060] The high sensitivity of this system allows for cell cycle studies, as the amount of nucleic acid is representative of the cell cycle status. By measuring the changes in nucleic acid content, the effect of anti-cancer drugs on tumor cells can be investigated. This can be done at the single cell level, or the recovered cancer cells can be cultured and used for tumor tissue regeneration in vitro or in vivo.

[0061] Figures 14a and 14b show the results of drug response analysis of MCF-7 cells. As a first step, cell viability was measured before and after analyzing these cells using any version of the device described in this disclosure. Figure 14a(a) clearly shows that viability was maintained and the cells remained intact. To further prove this phenomenon, cells taken after analysis were cultured for 3 days and proliferated like healthy cells. Figure 14a(b) shows an image of a well with cells immediately after analysis, and 14a(c) shows an image of the same well after 3 days of culture. STS (concentration 1 μM) was then added to this well and the cells were cultured under drug exposure for another 3 days.

[0062] Figure 14b shows the results of the drug response analysis, where cells were harvested from the wells and analyzed using the same instrument that was used to generate the reference data in the first place. In parallel, control samples, cultured under the same conditions but without drug exposure, were also analyzed for comparison. Figure 14b(a) shows the opacity distribution of the control and treated samples relative to one frequency, and Figure 14b(b) shows three clusters within the population. In the control sample, only one cluster was identified, viable cells, with a ratio of 97% of the total number of events. In the treated sample, three clusters were identified: viable, early apoptotic, and late apoptotic, with ratios of 44%, 19%, and 37%, respectively. For validation, a commercially available apoptosis detection kit for fluorescent imaging was used, which showed a viable rate of 47% and an apoptotic rate of 53% in the treated samples. The high-resolution drug response analysis was performed in a completely label-free manner, leaving the cells intact.

[0063] The present disclosure improves the sensitivity and signal-to-noise ratio. Therefore, the magnitude of the voltage required to generate the excitation and measurement signals is much lower than in the state of the art. This is an essential requirement to avoid electroporation, which is the formation of holes on the cell membrane due to the voltage drop caused by charging the cell membrane acting like a capacitor. In particular, the detection unit used is structured to detect the analyte in the sample liquid with a voltage drop between the electrodes in the range of 0.1-0.5 V, preferably 0.2 V-0.4 V.

[0064] Pore ​​formation begins in most mammalian cells when the voltage drop on the cell membrane is greater than 0.2 V. Pore formation is reversible up to 1 V-1.5 V and becomes an irreversible process above this threshold. Electroporation induces changes in the biological, morphological properties and integrity of the cell. This voltage level is harmful to living cells, since it exceeds the electroporation limit for most cells. The present disclosure, offering high sensitivity and low noise, allows for successful detection and characterization of cells at voltages below this electroporation limit, thus maintaining viability and keeping the analyzed cells intact (Figure 14a).

[0065] In the present disclosure, measurement results are obtained when the electric field generating unit in the present disclosure provides a voltage drop between the electrodes of the present disclosure in the range of 0.1V to 0.5V, preferably in the range of 0.2V to 0.4V. The results shown in Figures 12, 13 and 14 are obtained at voltage values ​​varying between 0.2V and 0.4V at frequencies between 1MHz and 400MHz, with the maximum voltage drop achievable being 0.12V, which is below the electroporation limit. In the present disclosure, there are no lower or upper limits on the applied voltage or frequency. These are determined based on the sample, the electrolyte used (moderate conductivity), the surrounding electronics (analyser, power supply, etc.), the size of the measurement volume, and the requirements of the analysis.

[0066] In addition to use as sensors, some embodiments of the present disclosure can be used for electrical processing and manipulation of cells and particles in suspension. The ability to control the electrode area in contact with the electrolyte by defining the coverage angle of the concave electrode provides a high level of control over the parameters applied for electrical processing and manipulation. Processing of particles and cells can include, but is not limited to, electroporation of cells for delivery of contents or extraction of intracellular contents after bioproduction. Pore formation during electroporation can be reversible or irreversible, and the process (electrode area, applied voltage, etc.) is well controlled and efficient with the proposed configuration.

[0067] Particle and cell manipulation can be achieved by, but is not limited to, generating dielectrophoretic forces on these particles and cells. These forces can be generated by targeting specific properties, such as size, morphology, or phenotype, and adjusting the frequency and amplitude of the excitation voltage based on the selected property. The dielectrophoretic forces can then be used to guide and direct the particles and cells in a flow, or to collect and focus them in defined regions within a device for downstream follow-up analysis.

Claims

1. 1. An apparatus for detecting an analyte, such as a cell, in a sample fluid, comprising: a fluid channel having a longitudinal channel axis and configured to allow a sample fluid containing an analyte to pass therethrough; At least one pair of electrodes; an electric field generating unit for generating an electric field between the at least one pair of electrodes; a detection unit for detecting an analyte in a sample fluid passing through the electric field; Equipped with each electrode of the at least one pair of electrodes has a three-dimensionally structured electrode surface; The device, wherein the electrode surface is curved relative to the longitudinal channel axis.

2. 2. The apparatus of claim 1, wherein the curved electrode surface of each electrode of the at least one pair of electrodes has a concave electrode surface.

3. 3. Apparatus according to claim 1 or 2, characterized in that the at least one electrode has an arc length between 10° and 135°, for example between 10° and 60°, in particular between 10° and 90°.

4. 4. The device of claim 1, wherein the fluid channel has a cross-section that comprises a circular shape.

5. 4. The device of claim 1, wherein the fluid channel has a cross-section that comprises an elliptical shape.

6. 6. The device according to claim 1, further comprising an optical sensing unit for optically sensing a sensing area of ​​the fluid channel between the at least one pair of electrodes.

7. the optical sensing unit has an optical sensing axis disposed at a mid-plane of the fluid channel; 7. The device of claim 6, wherein the optical sensing axis extends perpendicular to a longitudinal channel axis of the fluid channel.

8. The apparatus of claim 7 , wherein the optical sensing unit further comprises a photodetector positioned offset from a midplane of the fluid channel.

9. 9. Apparatus according to any of claims 6 to 8, characterized in that the optical sensing unit comprises a waveguide unit.

10. 9. Apparatus according to any one of claims 6 to 8, characterized in that the optical sensing unit comprises a laser device.

11. 11. The device according to claim 6, wherein the electrodes do not obstruct the optical path of a light-sensing unit for illumination and optical sensing.

12. A first substrate and a second substrate, 12. The device of claim 6, wherein the first substrate and the second substrate at least partially define a fluidic channel.

13. 13. The device of claim 1, further comprising at least one integrated waveguide unit optically coupled to a sensing region of the fluid channel between the at least one pair of electrodes.

14. 14. The device according to claim 13, characterized in that at least one waveguide unit is disposed between the first substrate and the second substrate and / or is integrated in the first substrate or the second substrate.

15. the first substrate has a first recess; the second substrate has a second recess; 15. The device of claim 12, wherein the first recess and the second recess at least partially form a fluid channel.

16. 16. The apparatus of claim 15, wherein the first recess and the second recess have the same shape.

17. 17. The apparatus of claim 15 or 16, wherein the first recess and the second recess each include an electrode disposed therein.

18. 18. A device according to any preceding claim, wherein the first electrode has substantially the same shape as the second electrode.

19. 19. An apparatus according to any preceding claim, wherein one or more of the electrodes has a surface with a cross-section that includes a circular shape.

20. 20. An apparatus according to any preceding claim, wherein one or more of the electrodes has a surface with a cross-section that includes an elliptical shape.

21. 21. An apparatus according to any one of claims 1 to 20, characterized in that the detection unit detects the analyte in the sample fluid with a voltage drop between the electrodes in the range of 0.1 to 0.5V, preferably 0.2V to 0.4V.

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